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Use of flagellins from the genus marinobacter as vaccination adjuvants

a flagellin and marinobacter technology, applied in the field of medicine, can solve the problems of inability to activate the immune system of bacteria flagellins, adverse secondary effects, and very susceptible birds and other animals to infection, and achieve the effects of low cross-reactivity, limited reactivity between flagellins, and limited cross-reactivity

Inactive Publication Date: 2013-08-08
INST NAT DE INVESTIGACION Y TECH AGRARIA Y ALIMENTARIA INIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes the use of two types of flagellins, from two different species, as vaccine adjuvants. These flagellins can be used alone or combined to help create a stronger immune response to peptides or proteins. The patent also explains how the vaccine adjuvants are made and how they can be used to improve the effectiveness of vaccines.

Problems solved by technology

On the other hand, it is also well-known that some bacterial flagellins are not capable of activating the immune system when a natural infection occurs, that is, they do not bind to the TLR5 receptor to induce the inflammatory response [12].
Although adjuvants improve the immune response, they may also cause adverse secondary effects, as in the case of Freund's complete adjuvant [16], [17], or even in the case of other adjuvants approved by the FDA or the EMEA.
Likewise, birds and other animals are very susceptible to infection by enteric Salmonella [29].
However, prior immunity against bacteria from the genus Marinobacter is unlikely, since it is a marine bacterium that is not known to infect human beings or other organisms, and the likelihood of contact with non-marine organisms to produce an immunological response therein is very low.

Method used

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  • Use of flagellins from the genus marinobacter as vaccination adjuvants
  • Use of flagellins from the genus marinobacter as vaccination adjuvants
  • Use of flagellins from the genus marinobacter as vaccination adjuvants

Examples

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example 1

Obtainment and Purification of the Recombinant Flagellin Sequences

[0079]In this invention, we study the functionality and the adjuvant-vaccinal capacity of Marinobacter algicola flagellins F and FR (DG893T strain). In order to compare the activity and the function of the Marinobacter flagellins, we have also obtained and used Salmonella typhimurium flagellin STF.

[0080]The F and FR genes of the Marinobacter algicola flagellins were chemically synthesised (MrGene, Germany) from the primary DNA sequences of both flagellins, SEQ ID NO: 19 and SEQ ID NO: 21, respectively. Subsequently, a sequence that encodes a histidine tail was added to said sequences (in order to facilitate the purification of the recombinant protein obtained) and a sequence that encodes for the KDEL peptide was also added (it prevents the degradation of the recombinant protein obtained). On the other hand, starting from the Salmonella typhimurium DNA (GenBank: AY649721), the flagellin gene was obtained by polymerase ...

example 2

In Vitro Study of the Activity of Marinobacter Flagellins F and FR

[0085]In order to analyse the in vitro activity of the recombinant Marinobacter flagellins F and FR obtained using the commercial “Bac-to-Bac® Baculovirus system” method (Invitrogen, USA), human CACO-2 colon carcinoma cells were used which constitutively express the TLR5 receptor. When TLR5 is activated in these cells, it induces the activation of MyD88 in order to finally secrete IL-8 to the extracellular medium [8-10].

[0086]CACO-2 cells (0.5 ml) were cultured in a 24-well plate (Nunc, Denmark) with an 80% confluence and a concentration of 5×105 cells / well. Subsequently, the recombinant proteins from flagellin STF (Salmonella typhimurium), used as a positive control, F and FR (Marinobacter algicola) at a final concentration of 1 μg / ml, were added to the culture medium, and incubated for 18 hours at 37° C. in a 5% CO2 atmosphere. Samples whereto nothing was added (MOCK) and others whereto PBS was added were used as ne...

example 3

“In Vivo” Study of the Vaccinal Capacity and the Immunogenicity of Marinobacter Fusion Flagellins F and FR Bound to 4 Tandem Copies of the DUD Epitope

[0088]In order to analyse in vivo the vaccinal capacity and the immunogenicity of the Marinobacter flagellins F and FR obtained using the commercial “Bac-to-Bac® Baculovirus system” method (Invitrogen, USA), groups of 5 mice were immunised, by subcutaneous route (sc), with 30 μg of purified fusion flagellins STF4DUD (Salmonella typhimurium), F4DUD and FR4DUD (Marinobacter algicola). A group of 5 mice were also immunised by intranasal route (in) with the F4DUD protein. As a control group, 5 mice were inoculated with the DUD tetrapeptide (4DUD). 3 immunisations without adjuvants were applied on days 0, 15 and 30 of the assay, and blood samples were obtained from all the animals prior to each immunisation and 15 days following the last immunisation, although the serological studies of the presence of antibodies were performed with the ser...

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Abstract

Use of flagellins from the genus Marinobacter as vaccine adjuvants. This invention is based on the use of two recombinant flagellins, F and FR, from the species Marinobacter algicola (DG893T strain), as vaccine adjuvants capable of developing a specific immune response, against peptides or proteins fused to said flagellins, or administered unfused jointly with the flagellins. This invention also describes new combined vaccination strategies, based on the two Marinobacter algicola flagellins and the Salmonella typhimurium flagellin.

Description

FIELD OF THE INVENTION[0001]This invention may be ascribed to the technical field of medicine, preferably within the branch of immunology, as well as the pharmaceutical industry, specifically the technological field of vaccine development.[0002]This invention relates to the use of recombinant bacterial flagellins from the species Marinobacter algicola as vaccine adjuvants capable of developing a specific immune response against peptides or proteins, fused or unfused, toward said flagellins. This invention also relates to vaccines that combine bacterial flagellins from the species Marinobacter algicola and bacterial flagellins from the species Salmonella typhimurium. BACKGROUND[0003]Flagellin is the generic name for the main structural protein that makes up bacterial flagella. They are cylindrical structures of variable length (approx. 530 nm) and about 21 nm in diameter [1]. The flagella are observed in both Gram-positive and Gram-negative bacteria; they are structures of variable l...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/39C07K14/195
CPCA61K39/0275A61K39/145A61K39/39A61K2039/55516C07K14/195A61K2039/6068C12N15/86C12N2710/14043C12N2760/16134A61K2039/543A61K2039/545C07K14/255A61K39/12Y02A50/30
Inventor GOMEZ CASADO, EDUARDO
Owner INST NAT DE INVESTIGACION Y TECH AGRARIA Y ALIMENTARIA INIA
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